1. Field of the Invention
This invention relates to DNA fragments containing an alkaline cellulase K gene derived from a bacterial strain which belongs to the genus Bacillus and is alkalophilic, i.e. capable of optimal growth in the alkaline pH region, to plasmids with said DNA fragments inserted therein, and further to transformant microorganisms transformed with such recombinant plasmids.
2. Description of the Prior Art
So far cellulase has been recognized as a group of complicated enzymes catalyzing those enzymatic reactions by which cellulose is degraded to glucose, cellobiose or cello-oligosaccharide. Cellulase is said to include those enzymes given the names of C.sub.1 enzyme, C.sub.x enzyme, .beta.-glucosidase, exo-.beta.-glucanase, endo-.beta.-glucanase, cellobiase and so forth according to the mechanisms of their action. For the past several decades studies on cellulase have searched for sources of cellulase supply among fungi, such as fungi of the genera Trichoderma, Aspergillus, Acremonium and Humicola, exclusively from the viewpoint of profitable utilization of biomass resources.
Recently, cellulase-containing detergent compositions for articles f clothing, for instance, are under development, where cellulase might find a novel industrial use. However, the great majority of cellulase species produced by microorganisms, inclusive of the above-mentioned fungi, are the so-called neutral or acid cellulase species which show optimal and stable enzymatic activity at neutral to acidic pH. The occurrence of the so-called alkaline cellulase which shows its highest activity and is stable at alkaline pH, hence can meet the requirements relative to detergent compositions for clothing items and is very rare. Thus, the methods so far known for the production of alkaline cellulase species having the possibility of their being used in detergent compositions for clothing items includes only the method of producing cellulase A which comprises cultivating an alkalophilic bacterial strain belonging to the genus Bacillus and recovering cellulase A from the culture medium (Japanese Patent Publication No. 50-28515), the method of producing alkaline cellulase 301-A which comprises cultivating a bacterial strain belonging to the genus Cellulomonas (Japanese Patent Application Laid-open No. 58-224686), the method of producing carboxymethyl cellulase which comprises cultivating the alkalophilic Bacillus strain No. 1139 [Fukumori, F., Kudo, T. and Horikoshi, K., J. Gen. Microbiol., 131, 3339 (1985)] and the method of producing alkaline cellulase which comprises using a strain of the genus Streptomyces (Japanese Patent Application Laid-open No. 61-19483). Meanwhile, very recently, it was found that Bacillus sp. KSM-635 (FERM BP-1485), a kind of alkalophilic bacteria, can produce alkaline cellulase K, which is suited for use in detergent compositions for clothing items, in significant quantities and, furthermore, its productivity was improved as a result of investigations on the method of cultivation. Thus the technology of industrial fermentative production of alkaline cellulase is making progress. On the other hand, even with this technology, the pH of the medium must be maintained on the alkaline side during cellulase fermentation and, in addition, further improvement in its potency is desired for realizing actual commercial production thereof.
Therefore, from the viewpoint of breeding by taking a genetic approach, it appears to be of very great significance to use gene manipulation techniques in an attempt to isolate the alkaline cellulase gene of an alkalophilic bacterial strain of the genus Bacillus, joining the resultant plasmid into a microorganism capable of growing under neutral conditions.